Golgi N-Glycosyltransferases Form Both Homo- and Heterodimeric Enzyme Complexes in Live Cells

Glycans (i.e. oligosaccharide chains attached to cellular proteins and lipids) are crucial for nearly all aspects of life, including the development of multicellular organisms. They come in multiple forms, and much of this diversity between molecules, cells, and tissues is generated by Golgi-resident glycosidases and glycosyltransferases. However, their exact mode of functioning in glycan processing is currently unclear. Here we investigate the supramolecular organization of the N-glycosylation pathway in live cells by utilizing the bimolecular fluorescence complementation approach. We show that all four N-glycosylation enzymes tested (β-1,2-N-acetylglucosaminyltransferase I, β-1,2-N-acetylglucosaminyltransferase II, 1,4-galactosyltransferase I, and α-2,6-sialyltransferase I) form Golgi-localized homodimers. Intriguingly, the same enzymes also formed two distinct and functionally relevant heterodimers between the medial Golgi enzymes β-1,2-N-acetylglucosaminyltransferase I and β-1,2-N-acetylglucosaminyltransferase II and the trans-Golgi enzymes 1,4-galactosyltransferase I and α-2,6-sialyltransferase I. Given their strict Golgi localization and sequential order of function, the two heterodimeric complexes are probably responsible for the processing and maturation of N-glycans in live cells.     

Fluorescence Resonance Energy Transfer (FRET) and Proximity Ligation Assays Reveal Functionally Relevant Homo- and Heteromeric Complexes among Hyaluronan Synthases HAS1, HAS2, and HAS3

In vertebrates, hyaluronan is produced in the plasma membrane from cytosolic UDP-sugar substrates by hyaluronan synthase 1–3 (HAS1–3) isoenzymes that transfer N-acetylglucosamine (GlcNAc) and glucuronic acid (GlcUA) in alternative positions in the growing polysaccharide chain during its simultaneous extrusion into the extracellular space. It has been shown that HAS2 immunoprecipitates contain functional HAS2 homomers and also heteromers with HAS3 (Karousou, E., Kamiryo, M., Skandalis, S. S., Ruusala, A., Asteriou, T., Passi, A., Yamashita, H., Hellman, U., Heldin, C. H., and Heldin, P. (2010) The activity of hyaluronan synthase 2 is regulated by dimerization and ubiquitination. J. Biol. Chem. 285, 23647–23654). Here we have systematically screened in live cells, potential interactions among the HAS isoenzymes using fluorescence resonance energy transfer (FRET) and flow cytometric quantification. We show that all HAS isoenzymes form homomeric and also heteromeric complexes with each other. The same complexes were detected both in Golgi apparatus and plasma membrane by using FRET microscopy and the acceptor photobleaching method. Proximity ligation assays with HAS antibodies confirmed the presence of HAS1-HAS2, HAS2-HAS2, and HAS2-HAS3 complexes between endogenously expressed HASs. C-terminal deletions revealed that the enzymes interact mainly via uncharacterized N-terminal 86-amino acid domain(s), but additional binding site(s) probably exist in their C-terminal parts. Of all the homomeric complexes HAS1 had the lowest and HAS3 the highest synthetic activity. Interestingly, HAS1 transfection reduced the synthesis of hyaluronan obtained by HAS2 and HAS3, suggesting functional cooperation between the isoenzymes. These data indicate a general tendency of HAS isoenzymes to form both homomeric and heteromeric complexes with potentially important functional consequences on hyaluronan synthesis.     

The High and Low Molecular Weight Forms of Hyaluronan Have Distinct Effects on CD44 Clustering

CD44 is a major cell surface receptor for the glycosaminoglycan hyaluronan (HA). Native high molecular weight hyaluronan (nHA) and oligosaccharides of hyaluronan (oHA) provoke distinct biological effects upon binding to CD44. Despite the importance of such interactions, however, the feature of binding with CD44 at the cell surface and the molecular basis for functional distinction between different sizes of HA is still unclear. In this study we investigated the effects of high and low molecular weight hyaluronan on CD44 clustering. For the first time, we provided direct evidence for a strong relationship between HA size and CD44 clustering in vivo. In CD44-transfected COS-7 cells, we showed that exogenous nHA stimulated CD44 clustering, which was disrupted by oHA. Moreover, naturally expressed CD44 was distributed into clusters due to abundantly expressed nHA in HK-2 cells (human renal proximal tubule cells) and BT549 cells (human breast cancer cell line) without exogenous stimulation. Our results suggest that native HA binding to CD44 selectively induces CD44 clustering, which could be inhibited by oHA. Finally, we demonstrated that HA regulates cell adhesion in a manner specifically dependent on its size. oHA promoted cell adhesion while nHA showed no effects. Our results might elucidate a molecular- and/or cellular-based mechanism for the diverse biological activities of nHA and oHA.     

Glycan-dependent and -independent Interactions Contribute to Cellular Substrate Recruitment by Calreticulin

Calreticulin is an endoplasmic reticulum chaperone with specificity for monoglucosylated glycoproteins. Calreticulin also inhibits precipitation of nonglycosylated proteins and thus contains generic protein-binding sites, but their location and contributions to substrate folding are unknown. We show that calreticulin binds glycosylated and nonglycosylated proteins with similar affinities but distinct interaction kinetics. Although both interactions involve the glycan-binding site or its vicinity, the arm-like proline-rich (P-) domain of calreticulin contributes to binding non/deglycosylated proteins. Correspondingly, ensemble FRET spectroscopy measurements indicate that glycosylated and nonglycosylated proteins induce “open” and “closed” P-domain conformations, respectively. The co-chaperone ERp57 influences substrate-binding kinetics and induces a closed P-domain conformation. Together with analysis of the interactions of calreticulin with cellular proteins, these findings indicate that the recruitment of monoglucosylated proteins to calreticulin is kinetically driven, whereas the P-domain and co-chaperone contribute to stable substrate binding. Substrate sequestration in the cleft between the glycan-binding site and P-domain is a likely mechanism for calreticulin-assisted protein folding.     

A Direct Interaction between the Sigma-1 Receptor and the hERG Voltage-gated K+ Channel Revealed by Atomic Force Microscopy and Homogeneous Time-resolved Fluorescence (HTRF?)

The sigma-1 receptor is an endoplasmic reticulum chaperone protein, widely expressed in central and peripheral tissues, which can translocate to the plasma membrane and modulate the function of various ion channels. The human ether-à-go-go-related gene encodes hERG, a cardiac voltage-gated K⁺ channel that is abnormally expressed in many human cancers and is known to interact functionally with the sigma-1 receptor. Our aim was to investigate the nature of the interaction between the sigma-1 receptor and hERG. We show that the two proteins can be co-isolated from a detergent extract of stably transfected HEK-293 cells, consistent with a direct interaction between them. Atomic force microscopy imaging of the isolated protein confirmed the direct binding of the sigma-1 receptor to hERG monomers, dimers, and tetramers. hERG dimers and tetramers became both singly and doubly decorated by sigma-1 receptors; however, hERG monomers were only singly decorated. The distribution of angles between pairs of sigma-1 receptors bound to hERG tetramers had two peaks, at ∼90 and ∼180° in a ratio of ∼2:1, indicating that the sigma-1 receptor interacts with hERG with 4-fold symmetry. Homogeneous time-resolved fluorescence (HTRF®) allowed the detection of the interaction between the sigma-1 receptor and hERG within the plane of the plasma membrane. This interaction was resistant to sigma ligands, but was decreased in response to cholesterol depletion of the membrane. We suggest that the sigma-1 receptor may bind to hERG in the endoplasmic reticulum, aiding its assembly and trafficking to the plasma membrane.     

Interactions of perilipin-5 (Plin5) with adipose triglyceride lipase

Members of the perilipin family of lipid droplet scaffold proteins are thought to play important roles in tissue-specific regulation of triglyceride metabolism, but the mechanisms involved are not fully understood. Present results indicate that adipose triglyceride lipase (Atgl) interacts with perilipin-5 (Plin5) but not perilipin-1 (Plin1). Protein interaction assays in live cells and in situ binding experiments showed that Atgl and its protein activator, α-β-hydrolase domain-containing 5 (Abhd5), each bind Plin5. Surprisingly, competition experiments indicated that individual Plin5 molecules bind Atgl or Abhd5 but not both simultaneously. Thus, the ability of Plin5 to concentrate these proteins at droplet surfaces involves binding to different Plin5 molecules, possibly in an oligomeric complex. The association of Plin5-Abhd5 complexes on lipid droplet surfaces was more stable than Plin5-Atgl complexes, and oleic acid treatment selectively promoted the interaction of Plin5 and Abhd5. Analysis of chimeric and mutant perilipin proteins demonstrated that amino acids 200-463 are necessary and sufficient to bind both Atgl and Abhd5 and that the C-terminal 64 amino acids of Plin5 are critical for the differential binding of Atgl to Plin5 and Plin1. Mutant Plin5 that binds Abhd5 but not Atgl was defective in preventing neutral lipid accumulation compared with wild type Plin5, indicating that the ability of Plin5 to concentrate these proteins on lipid droplets is critical to functional Atgl activity in cells.     

Architecture of Polyglutamine-containing Fibrils from Time-resolved Fluorescence Decay

The disease risk and age of onset of Huntington disease (HD) and nine other repeat disorders strongly depend on the expansion of CAG repeats encoding consecutive polyglutamines (polyQ) in the corresponding disease protein. PolyQ length-dependent misfolding and aggregation are the hallmarks of CAG pathologies. Despite intense effort, the overall structure of these aggregates remains poorly understood. Here, we used sensitive time-dependent fluorescent decay measurements to assess the architecture of mature fibrils of huntingtin (Htt) exon 1 implicated in HD pathology. Varying the position of the fluorescent labels in the Htt monomer with expanded 51Q (Htt51Q) and using structural models of putative fibril structures, we generated distance distributions between donors and acceptors covering all possible distances between the monomers or monomer dimensions within the polyQ amyloid fibril. Using Monte Carlo simulations, we systematically scanned all possible monomer conformations that fit the experimentally measured decay times. Monomers with four-stranded 51Q stretches organized into five-layered β-sheets with alternating N termini of the monomers perpendicular to the fibril axis gave the best fit to our data. Alternatively, the core structure of the polyQ fibrils might also be a zipper layer with antiparallel four-stranded stretches as this structure showed the next best fit. All other remaining arrangements are clearly excluded by the data. Furthermore, the assessed dimensions of the polyQ stretch of each monomer provide structural evidence for the observed polyQ length threshold in HD pathology. Our approach can be used to validate the effect of pharmacological substances that inhibit or alter amyloid growth and structure.     

Dimerization,Oligomerization, and Aggregation of Human Amyotrophic Lateral Sclerosis Copper/Zinc Superoxide Dismutase 1 Protein Mutant Forms in Live Cells

More than 100 copper/zinc superoxide dismutase 1 (SOD1) genetic mutations have been characterized. These mutations lead to the death of motor neurons in ALS. In its native form, the SOD1 protein is expressed as a homodimer in the cytosol. In vitro studies have shown that SOD1 mutations impair the dimerization kinetics of the protein, and in vivo studies have shown that SOD1 forms aggregates in patients with familial forms of ALS. In this study, we analyzed WT SOD1 and 9 mutant (mt) forms of the protein by non-invasive fluorescence techniques. Using microscopic techniques such as fluorescence resonance energy transfer, fluorescence complementation, image-based quantification, and fluorescence correlation spectroscopy, we studied SOD1 dimerization, oligomerization, and aggregation. Our results indicate that SOD1 mutations lead to an impairment in SOD1 dimerization and, subsequently, affect protein aggregation. We also show that SOD1 WT and mt proteins can dimerize. However, aggregates are predominantly composed of SOD1 mt proteins.     

Novel Zinc-binding Site in the E2 Domain Regulates Amyloid Precursor-like Protein 1 (APLP1) Oligomerization

The amyloid precursor protein (APP) and the APP-like proteins 1 and 2 (APLP1 and APLP2) are a family of multidomain transmembrane proteins possessing homo- and heterotypic contact sites in their ectodomains. We previously reported that divalent metal ions dictate the conformation of the extracellular APP E2 domain (Dahms, S. O., Könnig, I., Roeser, D., Gührs, K.-H., Mayer, M. C., Kaden, D., Multhaup, G., and Than, M. E. (2012) J. Mol. Biol. 416, 438–452), but unresolved is the nature and functional importance of metal ion binding to APLP1 and APLP2. We found here that zinc ions bound to APP and APLP1 E2 domains and mediated their oligomerization, whereas the APLP2 E2 domain interacted more weakly with zinc possessing a less surface-exposed zinc-binding site, and stayed monomeric. Copper ions bound to E2 domains of all three proteins. Fluorescence resonance energy transfer (FRET) analyses examined the effect of metal ion binding to APP and APLPs in the cellular context in real time. Zinc ions specifically induced APP and APLP1 oligomerization and forced APLP1 into multimeric clusters at the plasma membrane consistent with zinc concentrations in the blood and brain. The observed effects were mediated by a novel zinc-binding site within the APLP1 E2 domain as APLP1 deletion mutants revealed. Based upon its cellular localization and its dominant response to zinc ions, APLP1 is mainly affected by extracellular zinc among the APP family proteins. We conclude that zinc binding and APP/APLP oligomerization are intimately linked, and we propose that this represents a novel mechanism for regulating APP/APLP protein function at the molecular level.     

Characterization of the oligomeric structure of the Ca(2+)-activated Cl- channel Ano1/TMEM16A

Members of the Anoctamin (Ano)/TMEM16A family have recently been identified as essential subunits of the Ca²⁺-activated chloride channel (CaCC). For example, Ano1 is highly expressed in multiple tissues including airway epithelia, where it acts as an apical conduit for transepithelial Cl⁻ secretion and helps regulate lung liquid homeostasis and mucus clearance. However, little is known about the oligomerization of this protein in the plasma membrane. Thus, utilizing mCherry- and eGFP-tagged Ano1 constructs, we conducted biochemical and Förster resonance energy transfer (FRET)-based experiments to determine the quaternary structure of Ano1. FRET and co-immunoprecipitation studies revealed that tagged Ano1 subunits directly associated before they reached the plasma membrane. This association was not altered by changes in cytosolic Ca²⁺, suggesting that this is a fixed interaction. To determine the oligomeric structure of Ano1, we performed chemical cross-linking, non-denaturing PAGE, and electromobility shift assays, which revealed that Ano1 exists as a dimer. These data are the first to probe the quaternary structure of Ano1. Understanding the oligomeric nature of Ano1 is an essential step in the development of therapeutic drugs that could be useful in the treatment of cystic fibrosis.     

Development of Isoform-specific Sensors of Polypeptide GalNAc-transferase Activity

Humans express up to 20 isoforms of GalNAc-transferase (herein T1–T20) that localize to the Golgi apparatus and initiate O-glycosylation. Regulation of this enzyme family affects a vast array of proteins transiting the secretory pathway and diseases arise upon misregulation of specific isoforms. Surprisingly, molecular probes to monitor GalNAc-transferase activity are lacking and there exist no effective global or isoform-specific inhibitors. Here we describe the development of T2- and T3-isoform specific fluorescence sensors that traffic in the secretory pathway. Each sensor yielded little signal when glycosylated but was strongly activated in the absence of its glycosylation. Specificity of each sensor was assessed in HEK cells with either the T2 or T3 enzymes deleted. Although the sensors are based on specific substrates of the T2 and T3 enzymes, elements in or near the enzyme recognition sequence influenced their activity and required modification, which we carried out based on previous in vitro work. Significantly, the modified T2 and T3 sensors were activated only in cells lacking their corresponding isozymes. Thus, we have developed T2- and T3-specific sensors that will be valuable in both the study of GalNAc-transferase regulation and in high-throughput screening for potential therapeutic regulators of specific GalNAc-transferases.     

Vinculin Phosphorylation at Tyr1065 Regulates Vinculin Conformation and Tension Development in Airway Smooth Muscle Tissues

Vinculin localizes to membrane adhesion junctions in smooth muscle tissues, where its head domain binds to talin and its tail domain binds to filamentous actin, thus linking actin filaments to the extracellular matrix. Vinculin can assume a closed conformation, in which the head and tail domains bind to each other and mask the binding sites for actin and talin, and an open activated conformation that exposes the binding sites for talin and actin. Acetylcholine stimulation of tracheal smooth muscle tissues induces the recruitment of vinculin to the cell membrane and its interaction with talin and actin, which is required for active tension development. Vinculin phosphorylation at Tyr¹⁰⁶⁵ on its C terminus increases concurrently with tension development in tracheal smooth muscle tissues. In the present study, the role of vinculin phosphorylation at Tyr¹⁰⁶⁵ in regulating the conformation and function of vinculin during airway smooth muscle contraction was evaluated. Vinculin constructs with point mutations at Tyr¹⁰⁶⁵ (vinculin Y1065F and vinculin Y1065E) and vinculin conformation-sensitive FRET probes were expressed in smooth muscle tissues to determine how Tyr¹⁰⁶⁵ phosphorylation affects smooth muscle contraction and the conformation and cellular functions of vinculin. The results show that vinculin phosphorylation at tyrosine 1065 is required for normal tension generation in airway smooth muscle during contractile stimulation and that Tyr¹⁰⁶⁵ phosphorylation regulates the conformation and scaffolding activity of the vinculin molecule. We conclude that the phosphorylation of vinculin at tyrosine 1065 provides a mechanism for regulating the function of vinculin in airway smooth muscle in response to contractile stimulation.     

Hydrolysis Rates of Different Small Interfering RNAs (siRNAs) by the RNA Silencing Promoter Complex,C3PO,Determines Their Regulation by Phospholipase Cβ

C3PO plays a key role in promoting RNA-induced gene silencing. C3PO consists of two subunits of the endonuclease translin-associated factor X (TRAX) and six subunits of the nucleotide-binding protein translin. We have found that TRAX binds strongly to phospholipase Cβ (PLCβ), which transmits G protein signals from many hormones and sensory inputs. The association between PLCβ and TRAX is thought to underlie the ability of PLCβ to reverse gene silencing by small interfering RNAs. However, this reversal only occurs for some genes (e.g. GAPDH and LDH) but not others (e.g. Hsp90 and cyclophilin A). To understand this specificity, we carried out studies using fluorescence-based methods. In cells, we find that PLCβ, TRAX, and their complexes are identically distributed through the cytosol suggesting that selectivity is not due to large scale sequestration of either the free or complexed proteins. Using purified proteins, we find that PLCβ binds ∼5-fold more weakly to translin than to TRAX but ∼2-fold more strongly to C3PO. PLCβ does not alter TRAX-translin assembly to C3PO, and brightness studies suggest one PLCβ binds to one C3PO octamer without a change in the number of TRAX/translin molecules suggesting that PLCβ binds to an external site. Functionally, we find that C3PO hydrolyzes siRNA(GAPDH) at a faster rate than siRNA(Hsp90). However, when PLCβ is bound to C3PO, the hydrolysis rate of siRNA(GAPDH) becomes comparable with siRNA(Hsp90). Our results show that the selectivity of PLCβ toward certain genes lies in the rate at which the RNA is hydrolyzed by C3PO.     

Signal Recognition Particle-ribosome Binding Is Sensitive to Nascent Chain Length

The signal recognition particle (SRP) directs ribosome-nascent chain complexes (RNCs) displaying signal sequences to protein translocation channels in the plasma membrane of prokaryotes and endoplasmic reticulum of eukaryotes. It was initially proposed that SRP binds the signal sequence when it emerges from an RNC and that successful binding becomes impaired as translation extends the nascent chain, moving the signal sequence away from SRP on the ribosomal surface. Later studies drew this simple model into question, proposing that SRP binding is unaffected by nascent chain length. Here, we reinvestigate this issue using two novel and independent fluorescence resonance energy transfer assays. We show that the arrival and dissociation rates of SRP binding to RNCs vary according to nascent chain length, resulting in the highest affinity shortly after a functional signal sequence emerges from the ribosome. Moreover, we show that SRP binds RNCs in multiple and interconverting conformations, and that conversely, RNCs exist in two conformations distinguished by SRP interaction kinetics.     

Isozyme-specific Interaction of Protein Kinase Cδ with Mitochondria Dissected Using Live Cell Fluorescence Imaging

PKCδ signaling to mitochondria has been implicated in both mitochondrial apoptosis and metabolism. However, the mechanism by which PKCδ interacts with mitochondria is not well understood. Using FRET-based imaging, we show that PKCδ interacts with mitochondria by a novel and isozyme-specific mechanism distinct from its canonical recruitment to other membranes such as the plasma membrane or Golgi. Specifically, we show that PKCδ interacts with mitochondria following stimulation with phorbol esters or, in L6 myocytes, with insulin via a mechanism that requires two steps. In the first step, PKCδ translocates acutely to mitochondria by a mechanism that requires its C1A and C1B domains and a Leu-Asn sequence in its turn motif. In the second step, PKCδ is retained at mitochondria by a mechanism that depends on its C2 domain, a unique Glu residue in its activation loop, intrinsic catalytic activity, and the mitochondrial membrane potential. In contrast, of these determinants, only the C1B domain is required for the phorbol ester-stimulated translocation of PKCδ to other membranes. PKCδ also basally localizes to mitochondria and increases mitochondrial respiration via many of the same determinants that promote its agonist-evoked interaction. PKCδ localized to mitochondria has robust activity, as revealed by a FRET reporter of PKCδ-specific activity (δCKAR). These data support a model in which multiple determinants unique to PKCδ drive a specific interaction with mitochondria that promotes mitochondrial respiration.     

α-Hemoglobin Stabilizing Protein (AHSP), a Kinetic Scheme of the Action of a Human Mutant,AHSPV56G

A kinetic analysis has been made of the interaction of α-Hb chains with a mutant α-hemoglobin stabilizing protein, AHSP^V56G, which is the first case of an AHSP mutation associated with clinical symptoms of mild thalassemia syndrome. The chaperone AHSP is thought to protect nascent α chains until final binding to the partner β-Hb. Rather than protecting α chains, the mutant chaperone is partially unfolded but recovers its secondary structure via interaction with α-Hb. For both AHSP^WT and AHSP^V56G, the binding to α-Hb is quite rapid relative to the α-β reaction, as expected because the chaperone binding must be quite competitive to complete the α chain folding process before α-Hb binds irreversibly to β-Hb. The main kinetic difference is a dissociation rate of AHSP^V56G·α-Hb some four times faster relative to AHSP·α-Hb. Considering a role of protein folding, the AHSP^V56G apparently does not bind long enough (0.5 s versus 2 s for the WT) to complete the structural modifications. The overall replacement reaction (AHSP·α-Hb + β-Hb → AHSP + αβ) can be quite long, especially if there is an excess of AHSP relative to β-Hb monomers.     

Switch I closure simultaneously promotes strong binding to actin and ADP in smooth muscle myosin

The motor protein myosin uses energy derived from ATP hydrolysis to produce force and motion. Important conserved components (P-loop, switch I, and switch II) help propagate small conformational changes at the active site into large scale conformational changes in distal regions of the protein. Structural and biochemical studies have indicated that switch I may be directly responsible for the reciprocal opening and closing of the actin and nucleotide-binding pockets during the ATPase cycle, thereby aiding in the coordination of these important substrate-binding sites. Smooth muscle myosin has displayed the ability to simultaneously bind tightly to both actin and ADP, although it is unclear how both substrate-binding clefts could be closed if they are rigidly coupled to switch I. Here we use single tryptophan mutants of smooth muscle myosin to determine how conformational changes in switch I are correlated with structural changes in the nucleotide and actin-binding clefts in the presence of actin and ADP. Our results suggest that a closed switch I conformation in the strongly bound actomyosin-ADP complex is responsible for maintaining tight nucleotide binding despite an open nucleotide-binding pocket. This unique state is likely to be crucial for prolonged tension maintenance in smooth muscle.